| Propolis is a sticky and colloidal solid substance which consisted of resins collected by honeybees and secretion of bee gland.Studies on propolis in Xiaogan,Hubei,China have not been reported yet.Flavonoids are important bioactive compounds in propolis.They exhibit varieties of biological activities such as anti-oxidative,antimicrobial,anticancer and immunomodulatory effects.In recent years,the anticancer activity of propolis flavonoids has drawn increasing attention and become a research hotspot.However,most studies are concentrated on the activity of propolis flavonoids crude extracts or monomers such as galangin and chrysin.There are few reported researches on the antitumor activity of pinobanksin-3-acetate?PB3A?.The cytotoxicity of PB3A against liver cancer cells and its mechanism of action are not entirely clear.In this paper,the extraction,purification and antioxidant activity of propolis flavonoids as well as the antitumor activity of PB3A were mainly studied.This research can provide the scientific basis and theoretical guidance for the deep exploitation and utilization of propolis flavonoids resources.The main contents and results are as follows:1.Ultrasonic technique was employed for the extraction of total flavonoids from propolis in Xiaogan,Hubei.The effects of ultrasonic power,extraction time and alcohol concentration on the extraction yield of total flavonoids from propolis were investigated and the ultrasonic extraction condition was optimized by response surface methodology.The result showed that the optimum ultrasonic extraction condition was ultrasonic power of 150W,extraction time of 16 min and alcohol concentration of 86%,under which the extraction yield of propolis flavonoids was 21.10±0.075%?n=3?.2.Eight flavonoids and two phenolic acids were identified from propolis ethanolic extracts using LC-MS technique.These compounds were quercetin?m/z 301[M-H]-?,isorhamnetin?m/z 315[M-H]-?,kaempferol?m/z 285[M-H]-?,galangin?m/z 269[M-H]-?,chrysin?m/z 253[M-H]-?,pinocembrin?m/z 255[M-H]-?,pinobanksin?m/z 271[M-H]-?,pinobanksin-3-acetate?m/z 313[M-H]-?,caffeic acid?m/z 179[M-H]-?and caffeic acid phenethyl ester?m/z 283[M-H]-?.3.The flavonoids from propolis were separated and purified by preparative liquid chromatography and then the isolated white crystals were analyzed by various analytical techniques for purity analysis and structure identification.The results indicated that the purity of white crystals?compound 1?was determined as 96.82%using liquid chromatography method and compound 1 was identified as pinobanksin-3-acetate through various technologies such as MS,UV,IR and NMR.4.The antioxidant activity of flavonoids from propolis was assessed by radical scavenging assay.The results showed that the scavenging effects of propolis flavonoids on DPPH,superoxide and ABTS free radical both increased with increasing the concentrations and the IC50 values of propolis flavonoids scavenging DPPH,superoxide and ABTS free radical were0.038,0.746 and 0.006 mg/mL respectively,which suggested that propolis flavonoids had certain scavenging capacity on DPPH,superoxide and ABTS free radical.Therefore,the results proved that propolis flavonoids possessed the antioxidant activity.5.The cytotoxicity of PB3A against human tumor cell lines?HepG2,Hela and NCI-H460?and human normal liver cell?LO2?was evaluated by using cytotoxicity assay?MTT assay?.Furthermore,the effects of PB3A on Hep G2 cell migration and morphology were investigated by scratch test,AO/EB dual staining and Hoechst 33258 staining.Then,the mechanism of PB3A-induced Hep G2 cell apoptosis was determined by caspase-3 activity assay.The results of cytotoxicity assay showed that PB3A remarkably inhibited the growth of cancer cells?HepG2,Hela and NCI-H460?and the IC50 values of PB3A against HepG2,Hela and NCI-H460 cells were 79.2,133.5 and 148.3?M,respectively.Moreover,PB3A exhibited low cytotoxic activity toward LO2 cells with a IC50 value of 2245.4?M.These results suggested that PB3A exhibited significant cytotoxic activity against cancer cells?HepG2,Hela and NCI-H460?without damaging the normal LO2 cells.The result of scratch assay showed that the rate of cell migration decreased in the drug-treatment group,which indicated that PB3A could reduce migratory capacity of Hep G2 cells.The results of AO/EB dual staining and Hoechst 33258 staining suggested that cells in the drug-treatment group exhibited morphological changes such as nuclear DNA condensation,which proved that PB3A induced Hep G2 cell death by apoptosis.The result of caspase-3 activity assay showed that the caspase-3activity in HepG2 cells increased in the drug-treatment group,which demonstrated that PB3A might induce apoptosis in HepG2 cells through activation of caspase-3 pathway. |
PDF Full Text Request